Flow cytometric analysis of platelet surface antigens

Abstract

A flow cytometric analysis of human platelet surface antigens was carried out using a panel of monoclonal antibody reagents. The reagents used were specific either for the GPIb or the GP IIb/IIIa complex, surface immunoglobulin, or von Willebrand factor (vWf). Indirect surface immunophenotypes were determined using an EPICS V flow cytometer and the monoclonal antibodies 6D1, 10E5, Plt-1, UR1663, anti-IgG, and anti-vWf. Platelets were obtained from normal individuals or patients with either Bernard-Soulier syndrome (BSS) or Glanzmann's thrombasthenia (GT). Normal platelets were positive for 10E5, 6D1, Plt-1, and UR1663 and showed negligible activity for anti-IgG and anti-vWf. Platelets from individuals with BSS showed a ~ Normal platelet function depends upon the presence of a complex array of surface glycoprotein receptors and their interactions with vessel wall adhesive proteins. Platelet receptor-ligand interactions have been described for a variety of proteins including thrombin, von Willebrand factor (vWf), fibrinogen, thrombospondin, fibronectin, and agonists such as ADP, epinephrine, and collagen (25). The majority of these studies have been performed using radiolabeled ligands. The recent advent of hybridoma monoclonal antibody technology in conjunction with flow cytometry has resulted in the increased awareness of phenotypic complexity of leukocyte and lymphocyte subsets (13). Although there are few flow cytometric reports on platelets (1,10,17), these studies indicate that additional investigation might yield useful information. The application of flow cytometry (FCM) to the analysis of platelets should result in the rapid acquisition of single-cell multiparameter correlated data with high statistical precision. In our initial attempt to apply flow cytometry to platelets, a small panel of available platelet-specific monoclonal antibodies (McAb) was used to define conditions necessary for the flow cytometric analysis of platelet surface antigens. MATERIALS AND METHODS Platelet Isolation and Fluorescent StainingPeripheral venous blood (9 ml) was anticoagulated with 1 ml sodium citrate (13 mM) and EDTA (10 mM). marked reduction in 6D1, while the platelets of a patient with GT showed a marked reduction in binding of 10E5, Plt-1, and UR1663. Differences between histograms for normal platelets and for platelets from individuals with BSS or GT were evaluated using the Kolmogorov-Smirnov test. Compared to normal platelets, the BSS and GT platelets contain at least 35-fold less of the GPIb and G P IIb/IIIa complex respectively. Flow cytometry is a useful and precise method for the study of normal and abnormal surface platelet phenotypes.

Influence of Chloroquine or Acid Treatment of Human Platelets on the Antigenicity of HLA and the Thrombocyte-Specific' Glycoproteins la/lla, llb, and llb/llla

“…FC has been successfully used by Sugawara et al [5],Kurata et al [6] Marti et al [7], andMichelson [8]. In our study, the platelets remained unfixed during in cubation with the monoclonal antibodies and the secondstep reagents and during the wash procedures.…”

“…FC was performed according to the method of Marti et al [7], using a FACScan flow cytometer (Becton Dickinson) with standard equipment for the logarithmic measurement of the forward scatter signal (FSC), the 90°C side scatter signal (SSC), the green fluorscence (FL1), and the red fluorescence (FL2).…”

Calcium indirectly regulates immunochemical reactivity and functional activities of the N-domain of thrombospondin-1

“…10E5 is an integrin αIIbβ3 antibody (Coller et al, 1983) and was used as a positive control.T4 (Coulter) was used as an irrelevant antibody control. The percentage of positive cells was determined by flow cytometry versus a negative control without primary antibody (Marti et al, 1988).…”

A Flow-Cytometric Approach to Quantitative Estimation of Platelet Surface Immunoglobulin G

“…It has the unique advantage that, although cells are not visualised, measurements are made on individual cells, so that a phenotypic profile based on examination of a large cell population is obtained, enabling reliable de tection of subpopulations bearing different amounts of antigen. The main application of FC in haematology has been the study of white cell markers, but platelet FC is being increasingly used for the study of platelet mem brane glycoproteins [10][11][12][13], Detection of PSIg using FC has been the subject of several reports [14][15][16][17][18], and the general impression is that flow-cytometric methods are diagnostically useful in the setting of immune-mediated thrombocytopenia although an objective definition of their sensitivity in terms of numbers of IgG molecules detectable on the platelet surface is not available. This is because the results of PSIg estimation by FC are ex pressed either qualitatively or as arbitrary units based on fluorescence intensity, which makes comparisons be tween samples difficult, especially when, as usual, the flu orescent signals are scaled after passing through logarith mic amplifiers.…”